Micropropagation of mature Quercus ilex L. trees by axillary budding

This paper reports the successful micropropagation of mature Quercus ilex trees known as reluctant to in vitro propagation. Crown branch segments collected from 30 to 100 year-old trees were forced in order to promote the production of sprouting shoots that were used as a source of explants for initiating the cultures. Sterilization was critical and required low-level disinfestation protocols. Six out of the eight mature genotypes attempted were successfully inoculated and then maintained in culture with varying responses. Shoot proliferation of holm oak was influenced by BA concentration, with improved multiplication and shoot appearance when the BA concentration was sequentially reduced over the culture period. Micropropagation by axillary budding was achieved by culturing shoots on a sequence of cytokinin-enriched Lloyd and McCown (WPM) media alternating 2 week-long subcultures on 0.44 µM benzyadenine (BA) first, followed by 0.22 µM BA, then 0.044 µM BA plus 0.46 µM zeatin. Sucrose concentration and agar brand affected shoot proliferation, and the best results were obtained on WPM medium supplemented with 8 g L−1 Sigma agar (A-1296; Sigma-Aldrich) and 30 g L−1 sucrose. Addition of 20 µM silver thiosulphate had a significant positive effect on the appearance and development of shoots with a higher number of shoots being healthy and showing reduced shoot tip necrosis and early senescence of leaves. The 18.8% of the microshoots obtained for one clone could be rooted within 15 days on a half-strength Murashige and Skoog medium containing 14.8 µM or 24.6 µM indole-3-butyric acid and 0.54 µM α-naphthalene acetic acid.